The rhabdovirus virus, vesicular stomatic virus is a prototype negative strand RNA virus and other members of the Mononegavirales are serious human and animal pathogens. The aims of the proposal are to examine, both at the level of their primary nucleotide sequence and higher order structure, the cis- acting signals in the RNA genome of VSV that regulate individual steps in viral replication. Specifically it is proposed: 1. To analyze the role of primary RNA sequence and complementarily of the VSV genomic termini in controlling encapsidation, polymerase binding and RNA replication; 2. To analyze the role of primary RNA sequence and the leader RNA in influencing the decision of the polymerase at the leader-N junction to transcribe or replicate; 3. To define primary RNA sequence and higher-order structural elements at the intercistronic junctions that control the generation of discrete capped and polyadenylated mRNAs; 4. To analyze the cis-acting sequences required for assembly of replicated RNAs into infectious virus particles; 5. To analyze trans-acting factors in the control of transcription and replication; 6. To develop VSV as a vector for gene expression and delivery; 7. To perform high-resolution structural analyses of VSV N:P protein complexes, nucleocapsids and virions.